The present invention relates to the field of medicine and more precisely pertains to an immunomodulator exhibiting antimicrobial and antimycobacterial activities of the following formula: 
to a method for producing the same and to a pharmaceutical preparation for treating mycobacterioses as well as pulmonary chronic and nonspecific conditions, sexually transmitted diseases and the resulting immunodeficiency.
The antileprous preparation diuciphonum (SU author""s certificate No 459228) exhibits immunomodulating activities (SU author""s certificate No 938442) and has an immunostimulating effect on humoral and cellular immunity (V. P.Leskov et al., xe2x80x9cClinical pharmacology for physiciansxe2x80x9d, M., 1997, pp. 77-78; N. S. Prosorovsky, xe2x80x9cImmunologic effects of diuciphonum and utilization of the latter for analyzing a proliferative response of lymphacytesxe2x80x9d, synopsis of a thesis for a candidate""s degree in medicine, M., 1985; L. E. Kostyuk, xe2x80x9cImmunocorrecting properties of antileprous preparationsxe2x80x9d, synopsis of a thesis for a candidate""s degree in biology, 1986). However, this preparation lacks mitogenetic activity of its own and is capable to increase the proliferation of T-cells only in response to phytohemagglutinin (PHA). The toxicity of diuciphonum amounts to 2600 mg/kg.
It is known that, although exerting an antibacterial action, N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinsulfon)-Nxe2x80x2-isonicotinoylhydrazide (SU author""s sertificate No 768186) lacks immunotropic activity and, furthermore, it does not withstand prolonged storage conditions due to impurities contained therein. The immunomodulator exhibiting antimicrobial and antimycobacterial activities, the method for producing the same and the pharmaceutical preparation for treating secondary immunodeficiencies and resulting mycobacterioses which are claimed acording to the present invention are novel and have not been described in literature.
The problem taken as a starting point of the present invention is to provide a novel immunomodulator exhibiting antimicrobial and antimycobacterial activities, including the carriage of human immunodeficiancy virus (HIV), which immunomodulator is active not only in respect of mycobacteria of leprosy but also of those of tuberculosis as well as other deseases developing against the background of immunodeficiency, as well as to provide a method for producing this immunomodulator and a pharmaceutical preparation intended to be used at secondarty immunodeficiencies and mycobaterioses.
This problem is solved, in accordance with the present invention, by providing a novel immunomodulator exhibiting antimicrobial and antimycobacterial activities, this immunomodulator representing N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyridioxo-1,2,3,4-tetrahydro-5-pyrimidinsulfon)-Nxe2x80x2-isonicotinoylhydrazide hydrate of the following formula: 
There is also provided a method for producing a novel immunomodulator exhibiting antimicrobial and antimycobacterial activities, which immunomodulator represents N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinsulfon)-Nxe2x80x2-isonicotinoylhydrazide hydrate of the above formula.
According to the invention, the method of the present invention is carried out by reacting equimolecular amounts of 6-methyluracil-5-sulfochloride and isoniazide in acetonitrile at an elevated temperature. The pharmaceutical preparation for treating mycobacterioses, pulnonary chronic nonspecific conditions, sexually transmitted diseases and immunodeficiency, according to the present invention, comprises an immunomodulator exhibiting antimicrobial and antimycobacterial activities as the active substance, said immunomodulator representing N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinsulfon)-Nxe2x80x2-isonicotinoylhydrazide hydrate of the above formula, and a vehicle.
The preparation isophonum claimed according to the present invention can be administered per os in any suitable medical form and parenterally. This preparation contains the active substance in amounts of 0.05 to 0.3 g.
As was shown by clinical tests, isophonum normalizes the immune status of persons suffering from leprosy, tuberculosis, chronic nonspecific pneumonia and chlamydiosis by involving the person""s organism in the struggle against the disease; isophonum also stumulates the function of the cell by penetrating into it and carries the active substance as far as to the agent of disease and kills it. It is not ruled out that the preparation may have a direct effect on the sexually transmitted agents such as chlamydia, trichomonads, ureaplasmatic infection.
It should be stressed that isophonum is well tolerable, has no side effects and is compatible with modem antimycobacterial drugs (ethionamide, rifampicinum and others), this being supported by results of examination of leprous and tuberculous patients.
The toxicity of isophonum was determined on white unbred mice wheighing 18 to 20 g in an sharp experiment by Litchfild-Wilkokson""s procedure and was found to exceed 7000 mg/kg.
The compound of the present invention is a white, sometimes a yellowish-hued light crystalline powder which is difficult to dissolve in water or usual organic solvents. It is soluble in dimethylformamide, dimethylsulfoxide, deluted acids and alkalies; f. p.=255-256xc2x0 C. The structure of matter is confirmed by the data of PMR-spectroskopy.
The compound of the present invention and the pharmaceutical preparation on its basis were tested in an experiment carried out on animals and clinically on people.
Determination of immunotropic activity of isophonum was carried out. Immunotropic activity of isophonum was determined on a model of immunodeficiency caused by irradiation. To create an immunodeficient state the mice F1 (CBAxc3x97C57BL/6) were irradiated with gamma-rays on a radioterapeutic apparatus in a dose of 4 Gy (Gy is the irradiation unit gray) at an irradiation dose power of 0.55 Gr/min. The number of antibody-forming cells (AFC) in the spleens was calculated by Jeme-Nordin""s procedure. Isophonum and diuciphonum were introduced per os over a wide range of doses. The investigation results are represented in Table 1.
As shown in the table, isophonum is 1.5 to 2.5 times more effective than diuciphonum in all doses.
The influence on humoral immunity was studied using the method of localized-in-gel hemolysis. The hybrid mice F1 (CBA/C57BL6) were immunized by erythrocites of the drum in a dose of 5xc3x97106 and immediately after that the substances under study were administered per os in a starch suspenstion because the low solubility of isophonum does not permit introducing this compound intraperitoneally. On the 5th day the number of antibody-forming cells (AFC) in the spleen of the mice was calculated and the immune response stimulation index was determined as the ratio of AFC number in the test group to the AFC number in the control group. The respective data are represented in Table 2.
The immune response stimulation indices of isophonum and diuciphonum are reliably indistinguishable from each other. This points to the fact that both prepatations under study exert an equal action on the humoral immunity which is tested in the reaction of AFC accumulation in the spleen of mice.
The influence of the test compound on the proliferation of T-lymphocytes was studied on a model in vitro. To this end human mononucleator cells were preliminarily incubated during three hours in the presence of different concentrations of each test substance. Each test substance was studied over a wide range of its concentrations, that is from 0.01 to 5 xcexcg/kg. As a negative control there was added a physiological 0.9 percent solution of NaCl. The results of the influence of novel compoubds on the intensity of proliferation of human lymphocytes in the cell culture in vitro are represented in Table 3.
It follows that, firstly, isophonum possesses the proper mitogenetic activitiy of its own and, secondly, it is capable of enhancing the proliferation of T-cells in response to PHA (phytohemagglutinin). The optimal concentration is 0.1 xcexcg/ml.
The influence of the preparations in question on the phagocytic activity of macrophages was studid from clearence of the carcass in blood taken from the retroorbital sinus of mice that received the preparations per os. The results were estimated on the phagocytic index xcex1 (see Table 4).
From the results of this experiment it can be concluded that isophonum stimulates the phagocytic activity of macrophages by 56 percent.
Antituberculous activity of isophonum was tested versus the classical antituberculous preparation tubazide and the highly active immunomodulator diuciphonum. For the purpose of infecting the animals there were used laboratory ordinary and tubazide-resistent strains of tuberculosis mycobacteria H37RV up to 100 mg. 230 mice of the CBA line were infected intravenously with 0.25 mg of the culture of tuberculous micobacteria in 0.5 ml of physiological solution. The experiment lasted two months from the 10th of September to the 12th of November 1996. Observated were 16 groups of animals with 15 mice in each group. All the animals were kept under the same conditions of the vivarium on a standard diat.
During the course of the experiment the control groups of animals received 0.5 ml of a starch suspension through a probe five times a week. The test groups of animals received the preparations in different doses in 0.5 ml of a starch suspension. After the experiment all the animals, both the died and the killed, were weighed and so were their interior organs, the degree of damage to their interior organs was determined and, in addition, the action of the preparation was judged from the average life duration (LD). The results of the experiments are represented in Table 5.
As indicated by the above data, isophonum possesses a pronounced antituberculous action both on the mycobacteria that are sensitive to the most of antituberculous preparations and on the mycobacteria that are resistent to tubazide and streptomycin.
Antileprous activity of isophonum as compared with that of diuciphonum was studied on male mice of the CBA line infected with the laboratory Kyp-4 strain of leprosy mycobacteria taken in an amount of 5000 and introduced intraplantedly by Shepard""s procedure. The experiment lasted six months from the 7th of February to the 4th of June 1996. Observed were 5 groups of mice with 15 mice in each group. All the animals were kept under the same conditions of the vivarium on a standard diat.
The first group (controls): during six months of the experiment the mice received 0.5 ml of a starch suspension through a probe five times a week. After six months the animals were killed through a cervical dislocation of backbone, the organs were subjected to a bacterioscopic investigation, no pathologic deviations characteristic of any of the groups were found. The bacteriscopic investigation of the infected site was carried out by grinding the mouse foot in 2 ml of 0.1 percent solution of albumin, making a smear from 0.01 ml of the suspenstion and painting it by Ziel-Nielson""s procedure and counting the number of leprosy micobacteria in 60 fields of vision to determine the number of micobacteria per mouse in each group. The results of investigation are represented in Table 6.
As follows from the above data, isophonum in the dose studed (2.5 mg/kg) possesses bactericide activity against the leprosy mycobacteria.
Immunocorrecting and antimicrobial properties of isophonum revealed in the experiment were confimed by clinical testing of isophonum on persons suffering from leprosy (agent: mycobacteria of leprosy), on persons suffering from tuberculosis and pulmonary chronic nonspecific diseases (PCND) against the background of an immunodeficient state of the organism.
The pharmaceutical preparation of the present invention was studied clinically versus diuciphonum on people suffering from leprosy. The daily dose of the preparation was 0.6 g. Already after 3 to 4 weeks the patients who received isophonum felt an improvement of the general state of health with a decrease of inflammatory infiltrates on the skin and a change of their colour from rust-brown to pale pink. After 3 months the agent of leprosy disappeared in scrapes of nasal mucosa and the bacteriocscopic index darstically lowered. After 5 to 6 months the agent of leprosy could not be detected by bacterioscopic and histological investigation. The patients who received diuciphonum passed the same phases of clinical recovery, yet slower, because the agent of leprosy still continued to be detected by bacterioscopic and histological investigation in patients of this group some 10 to 12 months after. The results obtained are represented in Table 7.
The results of immunologic examination of the people suffering from leprosy who received treatment with isophonum and diuciphonum showed that both preparations normalize the status of the patient, i.e. both the T-lymphocytes and the T-helpers increase in number and the immunoregulatory index (ratio Th/Ts), which was lowered, is normalized. It shoud be noted that the normalization of immunologic parameters takes a smother course when isophonum is used, probably due to its affecting some other mechanisms of immunity than diuciphonum.
Thus, isophonum has shown a pronounced clinical antimycobacterial and immunotropic effect surpassing that of diuciphonum.
The use of isophonum enabled the length of stationary treatment of the leprous patients to be shortened two-fold.